Methods of angioplasty and cardiac catheterization

ABSTRACT

The present invention provides an improved mode of operation of an apparatus for angioplasty or cardiac catheterization, apparatus for angioplasty and cardiac catheterization, and method of angioplasty or cardiac catheterization. The improvement and improved mode of operation includes the step of administering a therapeutically effective amount of a lipid acceptor during angioplasty or cardiac catheterization of a subject with the apparatus or component thereof. The lipid acceptor is selected from the group consisting of a large liposome comprised of phospholipids substantially free of sterol and small acceptors. The effective period of time is in the range of about less than 1 minute to about two years from the time of the angioplasty or cardiac catheterization. The improved angioplasty or cardiac catheterization apparatus includes means for administering a therapeutically effective amount of a lipid acceptor, and optional co-administration means for administering the lipid acceptor and a diagnostic agent. The improved mode of operating an angioplasty or cardiac catheterization apparatus includes administering a therapeutically effective amount of a lipid acceptor from the apparatus or component thereof into a vessel of a subject by administration means. The invention further provides a method of diagnosing a side-effect of reverse transport of cholesterol from peripheral tissues to the liver in vivo accompanying parenteral administration of a multiplicity of large liposomes and small liposomes during a treatment period. The method includes the step of periodically assaying plasma atherogenic lipoprotein concentrations with an assay to obtain an assayed atherogenic lipoprotein concentration.

CONTINUING DATA

This application is a continuation in part regular patent application ofpending U.S. provisional patent application Ser. No. 60/005,090 filed byKevin Jon Williams, a citizen of the United States, residing at 425Wister Road, Wynnewood, Pa. 19096 on Oct. 11, 1995 entitled "METHOD OFFORCING THE REVERSE TRANSPORT OF CHOLESTEROL FROM PERIPHERAL TISSUES TOTHE LIVER IN VIVO WHILE CONTROLLING PLASMA LDL AND COMPOSITIONSTHEREFOR." Pending U.S. provisional patent application Ser. No.60/005,090 filed Oct. 11, 1995 is attached to the instant regular patentapplication as attachment A. Applicant expressly incorporates attachmentA hereto into the instant regular patent application by referencethereto as if fully set forth.

BACKGROUND OF THE INVENTION

Re-obstruction (re-stenosis) of arteries after mechanical or surgicalinterventions to improve blood flow is a major problem. For example,after coronary angioplasty, re-stenosis rates of 30-40% have beenreported. Re-stenosis after surgery to relieve carotid obstructions alsooccurs, and re-stenosis can occur in other vessels as well. Currenttreatments for re-stenosis include drugs, such as aspirin or lovastatin,that may somewhat reduce the incidence of restenosis; and the placementof mechanical stents in the vessel lumen to attempt to hold the vesselopen. Nevertheless, these methods are at best only partially effectiveand are often invasive. There exists a need for a simple, effective,non-invasive or minimally invasive approach to reduce re-stenosis orslow its development in patients who undergo mechanical or surgicalrevascularization procedures.

The process of re-stenosis involves, in part, the proliferation of cellsin the arterial wall in response to injury during the revasculariationprocedure. There is a role for cholesterol and possibly other lipids inthis process: high plasma concentrations of LDL and apolipoprotein B,the major protein of atherogenic lipoproteins, are associated withincreased carotid re-stenosis (Colyvas et al. Circulation 85:1286-1292,1992), and lovastatin, a cholesterol synthesis inhibitor, somewhatreduces coronary re-stenosis (Sahni et al Am. Heart J. 121 [6 pt 1]:1600-1608, 1996). There exists a need for better methods, devices, andmodes of operation of devices to manipulate the lipid content andcomposition of the arterial wall before, during, and afterrevascularization procedures, to reduce re-stenosis. Small LDL size hasalso been associated with increased re-stenosis (Colyvas 1992), and sothere exists a need for methods to change LDL composition and size.

The intravenous administration of cholesterol-poor phospholipid vesicles(liposomes) or other particles to transport cholesterol and otherexchangeable material from lipoproteins and peripheral tissues,including atherosclerotic arterial lesions, to the liver producessubstantial derangements in hepatic cholesterol homeostasis, such asenhanced hepatic secretion of apolipoprotein-B, and suppression ofhepatic LDL receptors. The hepatic derangements lead to increased plasmaconcentrations of LDL and other atherogenic lipoproteins. Increasedconcentrations of LDL or other atherogenic lipoproteins will accelerate,not retard, the development of vascular complications. Deranged hepaticcholesterol homeostasis can also be manifested by abnormal regulation ofother genes, such as a gene for the LDL receptor, a gene for HMG-CoAreductase, a gene for cholesterol 7-alpha hydroxylase, and a generegulating a function involved in cholesterol homeostasis. There existsa need for methods or compounds that can produce a removal ofcholesterol and other exchangeable material from peripheral cells,tissues, organs, and extracellular regions, but without harmfullydisrupting hepatic cholesterol homeostasis.

In general, several human conditions are characterized by distinctivelipid compositions of tissues, cells, or membranes. For example, inatherosclerosis, cholesterol (unesterified, esterified, and oxidizedforms) and other lipids accumulate in cells and in extracellular areasof the arterial wall and elsewhere. These lipids have potentiallyharmful biologic effects, for example, by changing cellular functionsand by narrowing the vessel lumen, obstructing the flow of blood.Removal of these lipids would provide numerous, substantial benefits. Inaging, cells have been shown to accumulate sphingomyelin andcholesterol, which alter cellular functions. These functions can berestored in vitro by removal of these lipids and replacement withphospholipid from liposomes. A major obstacle to performing similarlipid alterations in vivo has been disposition of the lipids mobilizedfrom tissues, cells, extracellular areas, and membranes. Natural (e.g.,high-density lipoproteins) and synthetic (e.g., small liposomes)particles that could mobilize peripheral tissue lipids have asubstantial disadvantage: they deliver their lipids to the liver in amanner that disturbs hepatic cholesterol homeostasis, resulting inelevations in plasma concentrations of harmful lipoproteins, such aslow-density lipoprotein (LDL), a major atherogenic lipoprotein.

The invention described herein provides methods and compositions relatedthe removal of cholesterol and other lipids from peripheral tissues, andotherwise altering peripheral tissue lipids, in patients undergoingrevascularization procedures, while controlling plasma concentrations ofLDL and other atherogenic lipoproteins and avoiding harmful disruptionof hepatic cholesterol homeostasis.

This invention methods and compositions that related to the "reverse"transport of lipids and other exchangeable material from peripheraltissues to the liver in vivo while controlling plasma LDLconcentrations. There exists a need for a method of, treatment, and apharmaceutical composition for forcing the reverse transport of lipidsfrom peripheral tissues to the liver in vivo while controlling plasmaLDL concentrations; of regulating hepatic parenchymal cell cholesterolcontent and metabolism in a cell having at least one gene selected fromthe group consisting of a gene for an LDL receptor, a gene for HMG-CoAreductase, a gene for cholesterol 7-alpha-hydroxylase, and a generegulating a function involved in cholesterol homeostasis; and,homeostasis thereof; suppressing hepatic expression of a cholesterolester transfer protein gene in vivo, whereby plasma LDL and HDL arecontrolled as a result of said administration; suppressing the rise inplasma LDL concentrations after administration of an agent having smallacceptors of cholesterol or other lipids; of diagnosing a side-effect ofreverse transport of cholesterol from peripheral tissues to the liver invivo accompanying parenteral administration of a multiplicity of largeliposomes and small liposomes during a treatment period, whereby a sideeffect of administration of said liposomes is diagnosed and effectivelyregulated; and, diagnosing and treating a side-effect of reversetransport of lipids from peripheral tissues to the liver in vivoaccompanying parenteral administration of a multiplicity of largeliposomes and small liposomes during a treatment period. There furtherexists a need for a system in which patients will have a decreased riskof developing atherosclerosis and/or cellular changes from aging; animproved method of reducing the lipid content of lesions. Other needsand solutions to these needs will be revealed further herein.

The present invention addresses these needs so that diseases anddetrimental medical conditions can be treated, controlled or eliminated.The present invention targets a market of millions of individualsworld-wide who suffer from medical conditions the present invention isdirected to solving. It is an object of the invention to solve theproblems enumerated above.

SUMMARY OF THE INVENTION

The present invention provides an improved mode of operation of anapparatus for angioplasty or cardiac catheterization, apparatus forangioplasty and cardiac catheterization, and method of angioplasty orcardiac catheterization. The improved mode of operation includes thestep of administering a therapeutically effective amount of a lipidacceptor during angioplasty or cardiac catheterization of a subject withthe apparatus or component thereof. The lipid acceptor is selected fromthe group consisting of a large liposome comprised of phospholipidssubstantially free of sterol and small acceptors. The effective periodof time is in the range of about less than 1 minute to about two yearsfrom the time of the angioplasty or cardiac catheterization. Theimproved angioplasty or cardiac catheterization apparatus includes meansfor administering a therapeutically effective amount of a lipidacceptor, and optional co-administration means for administering thelipid acceptor and a diagnostic agent. The improved mode of operating anangioplasty or cardiac catheterization apparatus includes administeringa therapeutically effective amount of a lipid acceptor from theapparatus or component thereof into a vessel of a subject byadministration means disposed on said apparatus. The invention furtherprovides a method of diagnosing a side-effect of reverse transport ofcholesterol from peripheral tissues to the liver in vivo accompanyingparenteral administration of a multiplicity of large liposomes and smallliposomes during a treatment period. The method includes the step ofperiodically assaying plasma atherogenic lipoprotein concentrations withan assay to obtain an assayed atherogenic lipoprotein concentration. Theobjects and features of the present invention other than thosespecifically set forth above, will become apparent in the detaileddescription of the invention.

It is an object of the invention to provide a simple, effective,non-invasive or minimally invasive approach, method, device, and mode ofoperation of the device to reduce re-stenosis or slow its development inpatients who undergo mechanical or surgical revascularizationprocedures.

It is a further object of the invention to provide a method, device, andmode of operation of a device to manipulate the lipid content andcomposition of the arterial wall before, during, and afterrevascularization procedures, to reduce re-stenosis. It is yet a furtherobject of the invention to provide for a method to change LDLcomposition and size.

It is yet another object of the invention to provide a method, compound,device and mode of operation of a device that can produce a removal ofcholesterol and other exchangeable material from peripheral cells,tissues, organs, and extracellular regions without harmfully disruptinghepatic cholesterol homeostasis. The objects and features of the presentinvention, other than those specifically set forth above, will becomeapparent in the detailed description of the invention set forth below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a side cross-sectional view of a lipoprotein and a liposome;

FIG. 2 illustrates a table of hepatic mRNA content (pg/μg) for CETP,HMG-CoAR, LDL receptors, and 7a-hydroxylase; and LDL ChE;

FIGS. 3 and 4 illustrate plasma LDL cholesteryl ester concentrations inresponse to injections of LUVs, SUVs or saline over time in one variant;

FIG. 5 illustrates LDL receptor mRNA levels in liver in response toinjections of LUVs, SUVs or saline over time;

FIG. 6 illustrates HMG-CoA reductase mRNA levels in liver in response toinjection of LUVs, SUVs, or saline;

FIG. 7 Illustrates cholesteryl ester transfer protein mRNA levels inliver in response to injection of LUVs, SUVs, or saline;

FIG. 8 illustrates 7-alpha hydroxylase mRNA levels in liver in responseto injections of LUVs, SUVs, or saline;

FIG. 9 illustrates key points about LUVs and atherosclerosis;

FIG. 10 illustrates plasma LDL unesterified cholesterol concentrationsin response to injections of LUVs, SUVs or saline over time;

FIG. 11 illustrates plasma LDL esterified cholesterol concentrations inresponse to injections of LUVs, SUVs or saline over time;

FIG. 12 illustrates LDL esterified cholesterol concentrations inresponse to injections of LUVs, SUVs or saline;

FIG. 13 illustrates plasma VLDL esterified cholesterol concentrations inresponse to injections of LUVs, SUVs or saline;

FIGS. 14 and 15 illustrate HDL esterified cholesterol concentrations inresponse to injections of LUVs, SUVs or saline;

FIG. 16 illustrates the time course of cholesterol mobilizationfollowing an LUV injection into control or apoE KO mice;

FIG. 17 illustrates the time course of LUV clearance in control mice andapoE mice;

FIG. 18 illustrates that the compositions and methods of the presentinvention are effective in humans;

FIG. 19 illustrates a perspective view of an improved hemodialysissystem of the present invention and improved method of hemodialysis;

FIG. 20 illustrates a perspective view of an improved peritonealdialysis system 2000 and method of peritoneal dialysis;

FIG. 21 illustrates a perspective view of a variant of an improvedperitoneal dialysis system with assaying means 2100 and method ofperitoneal dialysis and analysis of spent fluid;

FIG. 22 illustrates a perspective view of an improved cardiaccatheterization and/or angioplasty system 2200 and method of cardiaccatheterization and/or angioplasty;

FIG. 23 illustrates a perspective view of a variant of an improvedcardiac catheterization and/or angioplasty system 2300 and method ofcardiac catheterization and/or angioplasty;

FIG. 24 illustrates a graph of hepatic lipid contents in response toinjections of LUVs, SUVs, or saline;

FIG. 25 illustrates plasma free cholesterol concentrations followingrepeated injections of SUVs or LUV (300 mg/kg) in NZW rabbits;

FIG. 26 illustrates plasma cholesterol ester concentrations followingrepeated injections of SUVs or LUV (300 mg/kg) in NZW rabbits;

FIG. 27 illustrates alternations in plasma components after repeatedinjections of SUVs; and,

FIG. 28 illustrates an agarose gel electrophoresis of whole plasmafollowing repeated injections of LUVs, SUVs, or saline.

DETAILED DESCRIPTION OF THE INVENTION

FIG. 1 illustrates a schematic illustration of the structure of a normallipoprotein 100 and a unilamellar liposome 200. Lipoprotein 100 andliposome 200 are comprised of a phospholipid molecule 300. Phospholipidmolecules generally have polar head 500 and a fatty acyl chains 400.Molecule 600 represents a molecule of unesterifed cholesterol.Lipoprotein 100 is comprised of a hydrophobic core 102 composed mainlyof triglycerides and cholesteryl esters surrounded by a monolayer ofphospholipid molecules 300 with their fatty acyl side chains 400 facingthe hydrophobic core 102 and their polar heads 500 facing thesurrounding aqueous environment (not shown). Unesterified cholesterol600 is found largely within the phospholipid monolayer. Apolipoprotein700 is disposed within phospholipid molecules 300. Artificialtriglyceride emulsion particles have essentially identical structures,either with or without protein.

Liposome 200 is comprised of phospholipid molecules 300 forming aphospholipid bilayer, e.g. one lamella, either with or without protein,in which fatty acyl side chains 400 face each other, the polar headgroups 500 of the outer leaflet face outward to the surrounding aqueousenvironment (not shown), and the polar head groups 500 of the innerleaflet face inward to the aqueous core 202 of the particle 200.Depending on the composition of particle 200, phospholipid bilayers canhave a large capacity for unesterified cholesterol and otherexchangeable material and components thereof. As illustrated in FIG. 1there is no sterol. Typically, such liposomes can pick up unesterifiedcholesterol from other lipid bilayers, such as cell membranes, and fromlipoproteins. Liposomes also pick up proteins and donate phospholipidsand other exchangeable material and components thereof. Liposomes canalso have multilamellar structures, in which the bilayers are containedwithin the environment encapsulated by an outer bilayer to form multiplelamellae. The multiple lamellae can be arranged concentrically, like thelayers of an onion, or in another variant non-concentrically.

FIGS. 3 and 4 illustrate plasma LDL cholesteryl ester concentrations inresponse to injections of LUVs, SUVs or saline over time. Rabbits wereintravenously injected on days 1, 3 and 5 as indicated by arrows 302,304, and 306 respectively, with a bolus of 300 mg of phosphatidylcholineper kg of body weight or a matched volume of saline. Thephosphatidylcholine was pharmaceutical grade egg PC, in the form ofeither large unilamellar vesicles (LUVs) having diameters ofapproximately 100 NM (preferably ≅120 NM) prepared by extrusion (LUVswere measured at about 120 NM (123±35 NM and the extrusion membrane hadpores of about 100 NM in diameter) or small unilamellar vesicles withdiameters of approximately 30 NM (preferably 35 NM) prepared bysonication. (SUVs were measured in the range of 34±30 NM.) Blood wasdrawn just before each injection and on the sixth day at sacrifice.Plasma LDL cholesteryl ester concentrations were determined by a gelfiltration assay of the plasma with an in-line enzymatic assay forcholesteryl ester. Means ±SEMs are shown in FIG. 3. Animals infused withSUVs showed significantly higher plasma concentrations of LDLcholesteryl ester at days 3, 5, and 6 compared to either LUV-infused orsaline infused animals. FIGS. 2-8, 10-15, 24 and 28 illustrate data fromthe same experiment in which injections were made on days 1, 3, and 5and then livers were taken. Gel filtration was done of plasma to measurelipid contents of individual lipoprotein classes. FIG. 2 illustrates atable of hepatic mRNA content (pg/μg) for CETP, HMG-CoA R (hydroxymethylglutaryl coenzyme A reductase), LDL receptors, and cholesterol 7alpha-hydroxylase; and LDL ChE (low density lipoprotein cholesterylester) for the rabbits given saline (HEPES buffered saline) (rabbits1-4), LUVs (rabbits 5-8), and SUVs (rabbits 10-12) for the experimentdescribed for FIGS. 3 and 4. Rabbit 13 is the "Mix" rabbit.

FIG. 4 shows an animal labeled as mix. "Mix" refers to a single animalthat received SUVs on day 1, 3 and 5, but also one injection of LUVs onday 3. Before this injection of LUVs, the plasma concentration of LDLcholesteryl ester rose, but after the injection of LUVs, the LDLconcentration fell, despite continued injections of SUVs.

FIG. 5 illustrates LDL receptor mRNA levels in liver in response toinjections of LUVs, SUVs or saline over time. The rabbits describedabove were sacrificed at day 6, and samples of liver were snap-frozen inliquid nitrogen. mRNA was extracted, and rabbit mRNA for the LDLreceptor was quantified by an internal standard/RNase protection assay(Rea T. J. et al. J. Lipid Research 34:1901-1910, 1993 and Pape M. E.,Genet. Anal. 8:206-312, 1991). Means ±SEMs are shown in FIG. 5. Animalsinfused with SUVs showed significant suppression of hepatic LDL receptormRNA compared to LUV-infused or saline-infused animals. Suppression ofhepatic LDL receptor mRNA reflects parenchymal cell overload withsterol, and is a potentially harmful alteration from normal hepaticcholesterol homeostasis. In contrast, LUV-infused animals showed thehighest levels of hepatic LDL receptor mRNA, though the increase abovethat seen in the saline-infused animals did not reach statisticalsignificance. The liver from the "Mix" animal described above showed avalue of 5.28 pg LDL receptor mRNA/microgram which is closer to theaverage value in the saline group than in the SUV group. Thus, LDLreceptor mRNA was stimulated by the single injection of LUVs despiterepeated injections of SUVs.

FIG. 6 illustrates HMG-CoA reductase mRNA levels in liver in response toinjections of LUVs, SUVs, or saline. The experimental details are thoseas referenced above. Animals infused with SUVs showed significantsuppression of hepatic HMG-CoA reductase mRNA compared to LUV-infused orsaline infused animals. Suppression of hepatic HMG-CoA reductase mRNAreflects parenchymal cell overload with sterol, which can be apotentially harmful alteration from normal hepatic cholesterolhomeostasis. In contrast, LUV-infused animals showed the highest levelsof hepatic HMG-CoA reductase mRNA, though the increase above that seenin the saline-infused animals did not reach statistical significance.

The "mix" animal showed a value of 0.50 pg HMG-CoA reductasemRNA/microgram, which is essentially identical to the average value inthe saline group (0.51) and substantially higher than the value in theSUV group (0.27). Thus, HMG-CoA reductase mRNA was stimulated to itsnormal value by the single injection of LUVs, despite repeatedinjections of SUVs.

FIG. 7 illustrates cholesteryl ester transfer protein mRNA levels inliver in response to injection of LUVs, SUVs, or saline. Theexperimental details are those as referenced above.

Animals infused with LUVs showed significant suppression of hepatic CETPmRNA compared to SUV infused or saline infused animals. Suppression ofCETP mRNA produce changes in the plasma lipoprotein profile usuallyassociated with reduced risk of atherosclerosis. The "mix" animal showeda value of 3.18 pg CETP mRNA/microgram, which is closer to the averagevalue in the LUV group than in the SUV or saline groups. Thus, CETP mRNAwas suppressed by the single injection of LUV's despite repeatedinjections of SUVs.

FIG. 8 illustrates cholesterol 7-alpha hydroxylase mRNA levels in liverin response to injections of LUVs, SUVs, or saline. The experimentaldetails are those as reference above. Animals infused with SUVs showedsuppression of hepatic 7-alpha hydroxylase mRNA compared to LUV infusedor saline infused animals. Suppression of 7-alpha hydroxylase can be apotentially harmful alteration from normal hepatic homeostasis. Incontrast, LUV-infused animals showed the highest levels of hepatic7-alpha hydroxylase mRNA, though the increase above that seen in thesaline infused animals did not reach statistical significance. The "mix"animal showed a value of 0.51 pg 7-alpha hydroxylase mRNA/microgram,which is higher than the average value in the SUV group. Thus,7-alpha-hydroxylase mRNA was stimulated by the single injection of LUVs,despite repeated injections of SUVs.

FIG. 10 illustrates unesterified cholesterol concentrations in wholeplasma in response to injections of LUVs, SUVs, or saline over time. Theexperimental details are those as referenced above. As indicated by thisfigure, LUVs and SUVs significantly raised the plasma concentrations ofunesterfied cholesterol indicating mobilization of tissue stores. TheLUVs raised the concentration of unesterifed cholesterol more than didthe SUVs.

FIG. 11 illustrates esterified cholesterol concentrations in wholeplasma in response to injections of LUVs, SUVs or saline over time. Theexperimental details are those as referenced above. SUVs raised theplasma concentrations of cholesteryl ester on days 3, 5, and 6. FIG. 12duplicates the information contained in FIG. 3.

FIG. 13 illustrates plasma VLDL esterified cholesterol concentrations inresponse to injections of LUVs, SUVs, or saline. SUVs increased theplasma concentration of VLDL cholesteryl ester over that seen in thesaline of LUV treated groups. The "mix" animal showed a plasma VLDLcholesteryl ester concentration at day 6 of 2.4 mg/dl, which is lowerthan the average value in the SUV group. The experimental details arethose as referenced above.

FIGS. 14 and 15 illustrate HDL esterified cholesterol concentrations inresponse to injections of LUVs, SUVs, or saline. The experimentaldetails are those as referenced above as in FIG. 2. Suitablephospholipid can be obtained from Avanti Polar Lipids, Nippon Oil andFat in Japan and Princeton Lipids, as well as other suppliers. LUVs aremade through an extruder that is commercially available. SUVs caused asmall but statistically significant rise in HDL cholesteryl esterconcentrations on days five and six.

FIG. 16 illustrates the time course of cholesterol mobilizationfollowing an LUV injection into control or apoE KO (knock-out) micecommercially available from Jackson Laboratories, in Bar Harbor, Me.Control (C57/BL6) and apolipoprotein E knock-out mice were injected attime zero with a single bolus of 300 mg LUV phospholipid/kg body weight.The LUVs contained a tracer amount of labeled cholesterylhexadecylether, which remains on the liposomes after injection into amouse. Displayed data are for concentrations of total cholesterol, i.e.esterified plus unesterifed, in whole plasma. The rise in both sets ofanimals indicated that LUVs mobilize cholesterol into the plasma, evenin the presence of a severe genetic hyperlipidemia.

FIG. 17 illustrates the time course of LUV clearance in control mice andapoE mice. The experimental details are as described in FIG. 16. Theclearance of LUVs from the plasma is unimpaired in the apoE knock-outmice, indicating mobilization (FIG. 16) and disposal (FIG. 17) ofcholesterol even in the presence of a severe genetic hyperlipidemia.This indicates the usefulness of this preparation in hyperlipidemias.

FIG. 18 illustrates exemplary applications for the compositions andmethods of the present invention in humans. The therapeutic targets ofthe compositions and methods presented herein are lipid-rich, ruptureprone plaques, critical stenosis, post-angioplasty re-stenosis,atherosclerosis in general, and any membrane, cell, tissue, organ, andextracellular region and/or structure, in which compositional and/orfunctional modifications would be advantageous.

FIG. 19 illustrates a perspective view of an improved hemodialysissystem of the present invention and improved method of hemodialysis.Blood is taken from a site for circulatory access (shown here as arm1900) and transported into a cell-plasma separator 1910. The plasma isthen transported to a dialysis chamber 1920 and is divided into at leasttwo compartments that are separated by a semi-permeable membrane 1930.One side of the membrane 1930 is the patient's plasma 1940 and on theother side is the dialysate 1950. Selected molecules exchange across themembrane 1930 depending on the characteristics of the membrane (charge,pore size, etc.). The device 1960 comprises a device for adding lipidacceptors to the dialysate and for sampling the dialysate to allowassays of cholesterol, phospholipid, and other components, such asacceptors, specific lipoproteins, specific components, and to monitortreatment. Extraction of plasma cholesterol or other extractablematerial comprises several possibilities: 1) acceptors are disposed inthe dialysate that do not cross membrane 1930 into plasma; 2) theacceptors do cross membrane 1930 and are either left in the plasma andreturned to the patient or are separated from plasma before it isreturned to the patient; and/or 3) immobilized acceptors on a sheet(such as membrane 1930 itself), on beads, and/or on the walls of thechamber 1920. Plasma thus treated is returned to the patient, usuallyafter having being re-mixed with the blood cells. As noted, cholesterolacceptors can be added at any stage, as an example, a device 1970comprises acceptors and for adding acceptors to plasma shortly beforeits return into the patient is also illustrated in FIG. 19. It isfurther understood that contaminating cellular material, such asplatelets, in the plasma will also become cholesterol depleted inendogenous lipids and enriched in phospholipid. It is further understoodthat all acceptors mentioned throughout this application may acceptmolecules in addition to cholesterol and may donate material as well.

The cellular concentrate from the cell-plasma separator 1910 can then betreated in any of several ways before being returned to the patient: 1)returned to the patient with no further treatment (this includes beingmixed with plasma that has been treated as above); 2) transferred to asecond dialysis chamber (not shown) in which the dialysate containscholesterol acceptors to lipid deplete the cells of endogenous lipids,such as cholesterol, before their return to the patient; 3) mixed with asuspension or solution of lipid acceptors to lipid deplete the cells ofendogenous lipids, then either returned to the patient with theacceptors or option 1) and option 2) above can be performed with allcell types together, or after further separation into specific celltypes (for example, purified platelets could be lipid depleted ofendogenous lipids, such as cholesterol, and enriched in liposomallipids). Options 2) and 3) can be performed with periodic assays ofcellular cholesterol, phospholipid, fluidity, viscosity, fragility, cellcomposition and/or cell function. Devices 1960, 1970 include anapparatus that allows for the periodic sampling of cells duringtreatment. As with plasma, lipid acceptors can be added at any stage ofthe treatment. All fluids, e.g. plasma and concentrated cells, are movedby gravity, mechanically, by manual manipulation (a syringe), or withpumps as needed. Of course, it is understood that blood can be drawn forprocessing from any appropriate part of the body.

FIG. 20 illustrates a perspective view of an improved peritonealdialysis system 2000 and method of peritoneal dialysis. Patient'sabdomen 2010 (FIGS. 20-21) receives peritoneal dialysate 2020 stored incontainer 2030 into the peritoneal cavity through incision 2040 by wayof channel 2050. Lipid acceptors and/or cholesterol acceptors 2060 areoptionally disposed in container 2070. In another variant, lipidacceptors are added to dialysate 2020; added to container 2030 inconcentrated form shortly before infusion; added as shown to the streamof fluid entering the peritoneal cavity; or infused by a separate portalof entry into the patient by any effective route. Throughout thisapplication, it is understood that all acceptors may accept molecules inaddition to cholesterol and may donate material such as phospholipidsand antioxidants.

FIG. 21 illustrates a perspective view of a variant of an improvedperitoneal dialysis system with assaying means 2100 and method ofperitoneal dialysis and analysis of spent fluid. Container 2110 acceptsspent fluid from abdomen 2010 by way of channel 2120. The device 2110provides access to diagnostic samples of spent dialysate to allow forassay of cholesterol, phospholipid, and other parameters as describedherein showing the efficacy of the treatments described. Optionally,assay syringe 2130 is inserted by way of access portal 2140 into channelor tube 2120, or into container 2110, and optional pumps (not shown) areused to move the various fluids to appropriate locations for assaythereof.

FIG. 22 illustrates a perspective view of an improved cardiaccatheterization and/or angioplasty system 2200 and method of cardiaccatheterization and/or angioplasty. Patient 2210 undergoes cardiaccatherization and/or angioplasty. The patient intravenously receiveseffective doses of lipid acceptors or cholesterol acceptors 2230co-administered with said treatment(s) from container 2220.Intraarterial access of a catheter for coronary angiography and/orangioplasty allows for ready co-administration of cholesterol acceptorsand administration of diagnostic agents such as cholinergic agents, toassess vascular function.

FIG. 23 illustrates a perspective view of a variant of an improvedcardiac catheterization and/or angioplasty system 2300 and method ofcardiac catheterization and/or angioplasty. Catherization and/orangioplasty catheter 2310 has apertures 2320 that allow for the egressof cholesterol acceptors therefrom. In a variant, catheter 2310 has apermeable membrane that allow for the egress for cholesterol acceptorstherefrom. Phantom arrows 2330 indicate egress sites for cholesterolacceptors and/or diagnostic agents. Sites 2340 indicate entry sites forthe acceptors or agents. The balloon on the device 2300 can be replacedor supplemented with other devices or can form an inner balloon layerdisposed within an outer balloon layer. The acceptors are disposedbetween the inner and outer flexible balloon layers. Upon expansion ofsaid inner balloon layer a force is exerted against the fluid orgel-like acceptors forcing the acceptors out of the sites 2320, and intodirect contact (forcefully) against arterial lesions more locallydirecting the treatment. It will be appreciated that this variant of theinvention provides for maximal penetration of the acceptors into thearterial lesions. The infusions can be accomplished by gravity, manualmanipulation of a syringe, or by mechanical infusion pump 2350. The samemethod and system can be utilized with standard vascular imagingtechniques or vessels that include the femorals, carotids, andmesenteric vessels by way of example.

Patient 2210 undergoes cardiac catherization and/or angioplasty. Thepatient intravenously receives effective doses of cholesterol or lipidacceptors 2230 co-administered with said treatments(s) from container2220. Intraarterial access of a catheter for coronary angiography and/orangioplasty allows for ready co-administration of lipid or cholesterolacceptors and administration of diagnostic agents such as cholinergicagents, to assess vascular function.

Container 2110 accepts spent fluid from abdomen 2010 by way of channel2120. The device 2110 provides access to diagnostic samples of spentdialysate to allow for assay of cholesterol, phospholipid, and otherparameters as described herein showing the efficacy of the treatmentsdescribed. Optionally, assay syringe 2130 is inserted by way of accessportal 2140 into channel or tube 2120, and optional pumps (not shown)are used to move the various fluids to appropriate locations for assaythereof.

FIG. 24 illustrates a graph of hepatic lipid contents in response toinjections of LUVs, SUVs, or saline. The experimental details are asoutlined above. Liver samples were assayed for contents of severallipids: cholesterol ester (CE); triglyceride (TG); unesterifiedcholesterol (Chol); phosphatidylethanolamine (PE); andphosphatidylcholine (PC), which are displayed in units of μg(micrograms) lipid/mg. Lower values of PE and PC in the SUV-treatedanimals were produced; thus, the Chol:phospholipid ratios in theseanimals was higher than in the other groups.

FIG. 25 illustrates cholesterol ester concentrations following repeatedinjections of SUVs or LUVs (30 mg/kg) in NZW rabbits (New Zealand Whiterabbits). The arrows indicate times of phospholipid injection here ondays 0, 3 and 5. For a given phospholipid dose, LUVs promote a greaterrise in plasma free cholesterol concentrations.

FIG. 26 illustrates plasma free cholesterol concentrations followingrepeated injections of SUV or LUV (300 mg/kg) in NZW rabbits in the sameexperiment as in FIG. 25, arrows indicate times of phospholipidinjection. Repeated injections of LUV, unlike SUV, do not provoke adramatic rise in CE concentrations in plasma.

The rise in plasma CE concentrations that results from the delivery ofexcess cholesterol to the liver may be the consequence of two processes.It may involve an overproduction of CE-rich particles or an impairedclearance of CE-rich lipoproteins. Overproduction of CE-rich particlesthat occurs following SUV infusions may result in the plasma or in theliver. In plasma, LCAT acts on small unilamellar phospholipid vesiclesor on phospholipid enriched HDL generating CE which may be subsequentlytransferred by CETP onto LDL. The results with gel filtration of plasmafrom animals treated with SUVs indicate that CE is carried mostly orsubstantially on LDL. Also, in plasma, removal of apoE from VLDL by SUVswill slow the clearance of VLDL, thereby favoring a more efficientconversion into LDL. In the liver, the increased delivery of cholesterolto hepatocytes during cholesterol mobilization stimulates an oversecretion of apoB, CE-rich lipoproteins.

In a variant, the rise in plasma CE concentrations observed is theresult of an impaired clearance of CE rich atherogenic lipoproteins.Intravenously administered liposomes that acquire apoE compete with LDLfor LDL-receptor mediated uptake. The delivery of excess cholesterol tothe liver down regulates LDL receptors. The processes responsible for anincrease in plasma CE concentrations are different between the twoliposome preparations. LUVs, unlike SUVs, do not provoke a rise inplasma CE concentrations. LUVs are superior preparations for mobilizingtissue cholesterol without harmful side effects.

The method and composition of the present invention also providesenrichment of HDL cholesterol esters by SUVs. One contributing processis the stimulation of lecithin cholesterol acyl transferase (LCAT) andother processes related thereto. The ability of SUVs to increase HDLcholesterol ester is the result of stimulation of LCAT and otherprocesses related thereto. LCAT need phospholipid and cholesterol togenerate cholesteryl ester and lysophosphatidylcholine; liposomes cansupply extra phospholipid. The present invention also provides foralterations in lipoprotein (LDL, HDL, etc.) composition and function byLUVs and/or SUVs and/or other acceptors.

The liposome compositions described herein and methods utilizing samealso include the liposomes picking up endogenous apoE and hence blockingcellular uptake of LDL. The liposomes pick up apolipoproteins, such asapoE and apoA-I, and that this alters or enhances their functions. Forexample, the uptake of endogenous apoA-I enhances the ability ofliposomal derived phospholipid to pick up cholesterol, and the uptake ofendogenous apoE would allow the liposomes to block certain pathways forarterial uptake of lipoproteins. All of this is in the context ofcontrolling LDL levels and hepatic gene expression and cholesterolhomeostasis.

LUVs and SUVs deliver cholesterol to different regulatory pools withinthe liver. This conclusion is supported by the differences in hepaticgene responses and CETP mRNA is suppressed: the LDL receptor mRNA isunaffected or increased by LUVs but suppressed by SUVs; and CETP issuppressed by LUVs, but unaffected by SUVs. Further, it is understoodthat the arterial lesions referenced herein include, by way of example,critical stenoses.

The key points about LUVs and atherosclerosis are illustrated in FIG. 9.The practical benefits of using LUVs as a treatment for atherosclerosisare that they are straight forward to manufacture, and non-toxic even atvery high doses. Mechanistically, LUVs promote reverse cholesteroltransport in vivo without provoking a rise in LDL concentration, andLUVs are an optimal preparation.

The compositions that are used herein can direct clearance away fromhepatic parenchymal cells. And the various methods described herein areutilized with slow infusions of the compositions described, so thathepatic cells are not cholesterol overloaded even if clearance byparenchymal cells occurs. Further, HDL is also controlled by CETP genesuppression.

As described herein assays are performed by: assaying fasting plasmatriglyceride to estimate VLDL concentrations; assaying plasmacholesterol (free and ester, or total minus free=ester); precipitatingLDL (& VLDL) with polyanions-cations; assaying the supernatant which isHDL; and computing LDL's (whole plasma value minus VLDL-HDL) sterol (orsterol ester) in whole plasma. Liposomes will precipitate withpolyanions-cations; or optionally assaying the ester which liposomesmostly lack. Other assays include electrophoresis, chromatography,immune assays, electron microscopic assays, functional assays,structural assays, and compositional assays.

In the dialysate of the present invention, any liposomes or emulsionscould be used as long as it's a cholesterol acceptor and either it doesnot raise LDL or it is not returned to the patient's circulation. Ineither case, one would need to assay plasma LDL and the plasmaconcentration of the acceptors, and plasma concentrations of otheratherogenic lipoproteins.

With respect to the methods that require delivering the cholesterol tothe liver at a slow rate, or in low doses administration might permitsmall acceptors, such as SUVs, to be used without LUVs provided LDLlevels as levels of other atherogenic lipoproteins are monitored andregulated. To avoid disrupting hepatic cholesterol homeostasis, theentrapped drug as described herein need not be given at low doses, butrather the encapsulating liposome or emulsion is given in low doses; thedrug could be present at high amounts within a small number of liposomesor a small mass of liposomal lipid.

Alterations in HDL size, composition and function can be accomplished byadministering high or even truly low doses of large and/or smallliposomes that have little or no sterol. Liposomes without sterol, whengiven in low doses are easily broken apart by HDL and HDLapolipoproteins and then pieces are incorporated into the HDL fractionof plasma enriching it in phospholipid. Such small doses, e.g. 10-100mg/kg/dose, even of SUVs without LUVs or drugs to lower LDL levels, areunlikely to raise plasma LDL levels, although periodic monitoring wouldbe prudent.

Also, the method as disclosed herein of altering LDL composition withoutincreasing LDL concentration would be to enrich the composition withphospholipids, like POPC (palmitoyloleylphosphatidylcholine), that areresistant to oxidation, enrich the composition with anti-oxidants,deplete unesterified cholesterol, and reduce cellular or arterial uptakeof oxidized LDL by phospholipid enrichment.

Liposomes up to about 1000 NM or so would work in the present invention.Larger liposomes would also work but extraction of tissue lipoproteinmay be less efficient. It is further possible to concentrate or drycompositions of the present invention. These preparations are thendiluted or reconstituted at the time of therapy or administration. Inthis variant, a two component kit comprising the active material and adilutent is provided. Inclusion of phosphatidyl glycerol (PG) to makethe liposomes negatively charged, or charge other components of thecomposition, to prevent aggregation during storage is also provided.

FIG. 27 illustrates alterations in plasma components after repeatedinjections of SUVs. Watanabe Heritable Hyperlipidemic (WHHL) rabbitswere given intravenously 1000 mg of SUV phospholipid per kg of bodyweight, or the equivalent volume of saline, on Monday, Wednesday, &Friday of each week for three weeks (nine doses total). Three days afterthe final dose, blood samples were taken, and plasma components werefractionated by size by passage over a Superose-6 gel-filtration column.Eluents were read by an in-line spectrophotometer. The tracing on theright is from a saline-injected rabbit, and shows VLDL around fractions#17-18, and LDL around fraction #27. The tracing on the left is from anSUV-injected rabbit, and shows VLDL with persistent liposomes aroundfraction #16, and LDL-sized particles around fraction #25. The tracingsindicate an increase in the amount of LDL-sized particles after repeatedinjections of SUVs, consistent with an increase in LDL, which is aharmful effect. Because WHHL rabbits have a genetic lack of LDLreceptors, this result indicates that SUVs disrupt hepatic cholesterolhomeostasis not just by suppressing LDL receptor (FIG. 5), but also bymechanisms independent of LDL receptors (FIG. 27). LUVs avoid both LDLreceptor-dependent and independent disruptions.

FIG. 28 illustrates an agarose gel electrophoresis of whole plasmafollowing repeated injections of LUVs, SUVs, or saline. Experimentaldetails are referenced in FIGS. 2-8 & elsewhere herein. Four-μL plasmasamples from two rabbits in each group at day 6 were electrophoresedthrough 1% agarose then stained for lipids with Sudan black. O: origin.β: migration of an LDL standard. The SUV-mediated increase in LDLconcentration is illustrated by the darker but otherwise unremarkablei-band in those lanes. SUVs in plasma exhibited a mobility ahead of LDL,owing to their acquisition of plasma proteins, chiefly from HDL. Incontrast, plasma LUVs exhibited essentially the same mobility as freshlyprepared, protein-free vesicles, i.e., just above the origin (O),indicating a substantial absence or reduction of acquired proteins onthe LUVs.

Based on the electrophoretic mobilities in FIG. 28, quantification ofthe acquisition of protein by LUVs versus SUVs was obtained. LUVs andSUVs were incubated with human HDL in vitro for 4 hours at 37° C. thenseparated from the HDL by gel filtration chromatography and assayed forprotein and phospholipid. LUVs acquired 1.09 μg of protein per mg ofliposomal phospholipid, whereas SUVs acquired 40.4 μg/mg, i.e., almost40 times as much. Thus, the two types of liposomes exhibit a strikingquantitative difference in protein adsorption. SUVs, but not LUVs,avidly strip apoE from VLDL, thereby slowing its clearance from plasmaand favoring its conversion to LDL. In addition, adsorbed proteins playa role in directing the SUVs into a hepatic metabolic pool that disruptshepatic cholesterol homeostasis, whereas LUVs are not directed into sucha pool. Liposomes, emulsions, or any other particles or compounds thatextract tissue lipids but do not acquire large amounts of plasmaproteins behave similarly to LUVs in these regards.

Specific vascular genes affected by cholesterol loading of cells includegenes for prolyl-4-hydroxylase; hnRNP-K; osteopontin (there may be arole for oxidized lipids in provoking arterial calcifications); andMac-2. The methods of regulating these genes described herein effectrestoration of normal vascular or arterial function. Elevated expressionof prolyl-4-hydroxylase (an enzyme in the synthesis of collagen, acomponent of fibrotic plaques) and hnRNP-K (identified in pre-mRNAmetabolism and cell cycle progression) messages were found in aorticsmooth muscle cells after cholesterol feeding. These would normalizeafter the liposome treatments described herein. Other genes or enzymesthat are abnormal with cholesterol-loading and should normalize withliposome treatment as described herein include osteopontin, nitric oxidesynthase (NOS), adhesion molecules, chemoatractants, tissue factor,PAI-1 (plasmidigen activator inhibitor), tPA (tissue plasmidigenactivator) and Mac-2 (Ramaley et al. 1995). Other genes affected bycholesterol, cholesterol loading, oxidized lipids would also becorrected.

Many examples of small acceptors such as SUVs,apolipoprotein-phospholipid disks, and HDL are commercially availableand can be used in the invention. Kilsdonk EP et al. Cellularcholesterol efflux mediated by cyclodextrins, J. Biol. Chem.270:17250-17256, 1995. By way of further example, another small acceptorincludes the cyclodextrins. Small acceptors (specifically HDL) shuttlecholesterol from cells to liposomes. Cyclodextrins and also other smallacceptors can shuttle cholesterol and other exchangeable material fromcultured cells to LUVs, which substantially increases the removal anddonation of material between cells and LUVs.

Examples of anti-hyperlipidemic drugs include fibric acid derivatives,HmG CoA reductase inhibitors, Niacin, probucol, bile acid binders, otherdrugs and combinations thereof. Anti-hyperlipidemic treatments alsoinclude LDL, apheresis, ileal bypass, liver transplantation and genetherapy.

The data presented in this application support three possibleexplanations for the difference in metabolic response to LUVs versusSUVs. The three mechanisms act separately or in combination. First, LUVsare taken up largely by Kupffer cells, whereas SUVs are primarilydirected towards hepatic parenchymal cells. This is partly a mechanicalconsequence of hepatic architecture: hepatic endothelial fenestrae areoval openings of about 100×115 nm, through which SUVs of 30-nm diameteror so can readily pass and gain access to parenchymal cells. Largeparticles, such as large liposomes, of sufficient diameter will not passeasily, and are cleared instead by the macrophage Kupffer cells thatline the liver sinusoids. While SUVs also have access to Kupffer cells,their sheer number (˜10 times as many SUVs as LUVs per mg ofphospholipid) appears to saturate the reticuloendothelial system, and soparenchymal cells predominate in their clearance. Other methods todirect artificial particles away from parenchymal cells are alsoavailable, such as by changing the particle structure or composition,including charge and specific ligands for cell-specific binding.

Cholesterol clearance pathways mediated by parenchymal versus Kupffercells have distinct metabolic consequences. Direct delivery ofcholesterol to parenchymal cells by SUVs suppresses sterol-responsivemessages (FIGS. 5, 6, & 8). Delivery of cholesterol to Kupffer cells canbe followed by gradual transfer of lipid to parenchymal cells, forexample, via the extensions of Kupffer cells that reach down through thespace of Disse to make physical contact with parenchymal cells. The rateof sterol delivery to the parenchymal cells by transfer from Kupffercells can be slower than by direct uptake; the chemical form of thesterol may be altered by the Kupffer cells before transfer; there isother cell-cell communication; and, based on other pathways for lipidtransfer amongst liver cells, the process of transfer from Kupffer toparenchymal cells may be regulated, whereas SUV clearance does notappear to be.

The second contributing explanation for the difference in metabolicresponse to LUVs versus SUVs is based solely on differences in thekinetics of their delivery of cholesterol to the liver. LUVs are clearedfrom plasma somewhat more slowly than are SUVs, and thereby produce arelatively constant delivery of cholesterol mass to the liver from thetime of injection until the bulk of injected material is cleared. SUVsare cleared more rapidly, thereby delivering a large bolus ofcholesterol mass to the liver several hours after each injection, whichis followed by the sustained rise in plasma concentrations ofcholesteryl ester and atherogenic lipoproteins. The slow, steadydelivery by LUVs avoids disrupting hepatic cholesterol homeostasis,while the more rapid uptake of SUV cholesterol overwhelms the ability ofthe liver to maintain homeostasis, thereby provoking suppression ofhepatic LDL receptors. Other methods to deliver artificial particles ortheir components to the liver at a proper rate are also available, suchas by changing the particle structure or composition, including chargeand specific ligand for cell-specific binding.

The third contributing explanation is based on the striking quantitativedifference in protein adsorption between the two types of vesicles (FIG.28), which, in that particular experiment, was a result of theirdistinct surface curvatures. Thus, SUVs, but not LUVs, would avidlystrip apoE from VLDL, thereby showing its clearance from plasma andfavoring its conversion to LDL. SUVs that acquire apoE will compete withVLDL, LDL, and other particles for receptor mediated uptake by theliver. Also, adsorbed apoproteins can play a role in directingphospholipid vesicles to different hepatic metabolic pools. Othermethods to reduce protein uptake by artificial particles are alsoavailable, such as by changing the particle structure or composition,including charge and specific ligands for cell-specific binding.

Overall, given the observation that cholesteryl ester and LDLconcentrations do not increase after delivery of large amounts ofcholesterol and other exchangeable material to the liver by LUVs, it wasapparent that delivery was to a specific metabolic pool or pools withunique properties that do not increase plasma concentrations ofatherogenic lipoproteins or harmfully disturb hepatic cholesterolhomeostasis, including the regulation of genes and other functions.Thus, these inventions can be regarded in part as a unique deliverysystem that brings original particle components, such as phospholipid,plus material acquired by the particles, such as cholesterol, to aspecific delivery site for harmless disposal and other additionalbenefits. The delivery system with these characteristics will be usefulin any situation whatsoever in which control of hepatic cholesterolhomeostasis, hepatic phospholipid homeostasis, and hepatic metabolism ingeneral is advantageous.

For example, in a situation in which it is desirable to modifyerthyrocyte lipids, a straightforward approach would be to administerartificial particles that can donate and remove the appropriate lipids.If SUVs are used for this purpose, however, they will transportcholesterol and other material to the liver in a harmful manner, to thewrong pool and/or at the wrong rate, and this will cause increases inplasma concentrations of atherogenic lipoproteins, which is anundesirable side-effect that would preclude this approach. In contrast,the use of large liposomes or other particles with similar propertieswill result in the proper delivery of original and acquired material, tothe proper pool(s) at a proper rate, so that the desired effect(modification of erythrocyte lipids) can be achieved without harmfulincreases in plasma concentrations of atherogenic lipoproteins.

As another example, it can be desirable to modify infectious agents,such as bacteria, fungi, and viruses, using the compositions and methoddescribed herein. Administration of large liposomes or other particleswith similar properties will remove and donate exchangeable materials toand from these infectious agents, and then the administered particleswill be delivered to the proper pool(s), so that the desired effect canbe achieved without harmful increases in plasma concentrations ofatherogenic lipoproteins.

As another example, a valuable therapy may provoke an increase in plasmaconcentrations of atherogenic lipoproteins as an unwanted side-effect.Administration of large liposomes or other particles with similarproperties will alter this response through the delivery of lipids andother material to the proper hepatic metabolic pool. The data with the"Mix" animal provides a specific example of this effect (FIG. 4).

There are several mechanisms for affecting arterial uptake,accumulation, and retention of lipoproteins. Liposomes can pick up apoEfrom atherogenic lipoproteins, thereby reducing lipoprotein binding toarterial cells and also competing for binding to arterial cells.Finally, alterations in LDL size and/or composition affect its bindingto extracellular matrix and affect subsequent, harmful alterationswithin the arterial wall, for example, susceptibility to oxidation orenzymatic modifications.

The action or mode of operation of large acceptors, such as largeliposomes, can be aided by small acceptors, and vice-versa, and thisapplies to both endogenous (e.g., HDL) and exogenous (e.g.,apoprotein-phospholipid complexes) small acceptors. Large acceptorspenetrate poorly into the interstitial space and appear to inefficientlyapproach the cell surface under certain circumstances. These effectsimpede their uptake and donation of exchangeable material frommembranes, cells, tissues, organs, and extracellular regions andstructures. Small acceptors do penetrate well into the interstitialspace and are able to approach the cell surface, thereby allowingefficient uptake of exchangeable material. Small acceptors have majordisadvantages, however. They have a very limited capacity to acquire ordonate material (even though the initial rate of acquisition or donationis rapid, until their capacity becomes saturated) and, once they haveacquired material, they deliver it to the liver in a way that disruptshepatic cholesterol homeostasis.

Large acceptors and small acceptors together, however, synergisticallyovercome each other's drawbacks through at least three mechanisms.First, the large acceptors act as a sink (or supply) for exchangeablematerial, while the small acceptors act as a shuttle that siphonsmaterial from peripheral stores to the large acceptors and in the otherdirection. Thus, for example, the small acceptors penetrate tissue,acquire (and/or donate) material from the tissue, and their capacitybecomes at least partly saturated. They leave the tissue and encounterthe large acceptors in the plasma, at which point the small acceptorsare stripped of tissue lipids. The capacity of the small acceptors isthereby restored, so that when they return to the tissue, they canacquire (and/or donate) more material. This cycle can continue manytimes. Second, the large acceptors can re-model some small acceptors.For example, large acceptors can donate phospholipid to HDL, whichincreases the capacity of HDL acquire tissue cholesterol and othermaterial. Third, as noted elsewhere, the presence of large acceptors canblock or reduce the harmful disruptions in hepatic cholesterolhomeostasis caused by the small acceptors.

Large liposomes avoid raising plasma concentrations of atherogeniclipoproteins in general, not just LDL. This list includes alllipoproteins that contain apoliprotein B (apoB), such as LDL, IDL, VLDL,Lp(a), β-VLDL, and remnant lipoproteins.

Immune cells are also the targets for depletion using the methods andmodes of operation disclosed herein. It is understood thatadministration of an HMG-CoA reductase inhibitor, pravastatin, tocardiac transplant recipients reduced their natural-killer-cellcytotoxicity in vitro, reduced episodes of rejection accompanied byhemodynamic compromise, reduced coronary vasculopathy, reduced plasmaLDL levels (and increased HDL levels), and significantly enhancedone-year survival. The effect on survival was dramatic: in the controlgroup, 22% died in the first year, whereas only 6% died in thepravastatin-treated group.

Immunologic effects of HMG-CoA reductase inhibitors have been reportedin vitro. These reported immunologic effects include the regulation ofDNA in cycling cells, the inhibition of chemotaxis by monocytes, theregulation of natural-killer-cell cytotoxicity, and the inhibition ofantibody-dependent cellular cytotoxicity. Regulation of such inhibitorsresults from changes in circulating lipids or other effects and byutilization of the methods and modes of operation disclosed herein.

HMG-CoA reductase catalyzes an early step in cholesterol biosynthesisand is crucial in the synthesis of molecules besides cholesterol. Addingcholesterol to immune cells treated with HMG-CoA reductase inhibitorsdoes not restore function, although the addition of mevalonate does.Although this suggests that cholesterol depletion is not directlyresponsible for the immune effects, the use of liposomes or otheracceptors to remove cholesterol from cells increases endogenousconsumption of mevalonate, as the cells try to make more cholesterol. Toimpede the ability of the immune or other cells to make up theircholesterol loss by picking up more LDL or other lipoproteins, themethods and treatment described herein are also be done in conjunctionwith therapies to lower plasma cholesterol concentrations (includingHMG-CoA reductase inhibitors, fibric acids, niacin bile acid binders,LDL-pheresis, etc.).

These processes include enhancement of cholesterol removal and reductionof cholesterol influx. Levels of HDL, the apparent natural mediator ofcholesterol removal from peripheral cells, increased in a treated groupof patients, and LDL levels were deceased. The administration of HMG-CoAreductase inhibitors in vivo usually causes very tiny changes inreductase enzyme activity: cells simply make more enzyme to overcome thepresence of the inhibitor. They also make more LDL receptors (especiallyin the liver) and so LDL levels fall.

The invention further provides for additives to PD (peritoneal dialysissolutions) that reduce the accelerated atherosclerosis that occurs inrenal failure.

Chemotaxis of monocytes is an important early event in atheroscleroticlesion development: monocytes become attracted to abnormal arteriallipid deposits, and to cellular products made in response to thepresence of these deposits, enter the vessel wall, transform intomacrophages, internalize the lipid by phagocytosis and/ole endocytosis,and become a major component of the so-called lipid-rich foam cells ofhuman atherosclerotic lesions. Thus, inhibition of monocyte chemotaxisis important for atherosclerosis as well and can be accomplished usingthe methods disclosed herein. Both cellular and humoral immunity seem tobe affected by reductase inhibition: cardiac rejection accompanied byhemodynamic compromise has often been associated with humoral rejection(i.e., that occurring without producing marked lymphocytic infiltrationin endomyocardial-biopsy specimens).

Pravastatin may interact with cyclosporine [an importantimmunosuppressive drug], which blocks the synthesis of interleukin-2 instimulated T-lymphocytes. The addition of interleukin-2 restored thenatural-killer-cell cytotoxicity and partly restored theantibody-dependent cytotoxicity that were inhibited inlovastatin-treated in vitro cell cultures. A synergy betweencyclosporine and pravastatin explains increased immunosuppression inrecipients of cardiac transplants, whereas patients without transplantswho receive HMG-CoA reductase inhibitors for hypercholesterolemia do nothave clinical immunosuppression.

Thus, the use of safe cholesterol acceptors with otherimmunosuppressives, such as cyclosporine &/or glucocorticoids (which canalso suppress IL-2) is also contemplated by this invention.

It is also appreciated that the invention utilizes derivatives ofvarious compounds described herein.

Pathological specimens from patients with cardiac transplants who havesevere coronary vasculopathy have been reported to have a highcholesterol content. Therefore, early cholesterol lowering withpravastatin may play a part in decreasing the incorporation ofcholesterol into the coronary arteries of the donor heart. Largeliposomes or other cholesterol acceptors are used to accomplish the sameeffect, quickly and directly, alone or in combination, therewith.

Immune modulations is important in many conditions, not just cardiactransplantation. Areas in which the above approaches could be used alsoinclude transplantations of other organs, autoimmune diseases (in whichthe body's immune system mistakenly attacks the body's own tissues),some infections (in which the immune reaction becomes harmful), and anyother situation in which immune modulation would be helpful.

With respect to infections, modification of the lipid content andcomposition of foreign objects in the body (such as infectious agents)while maintaining normal hepatic cholesterol homeostasis should also bementioned.

Oxidized lipids alter tissue function and cause damage, includingdecreased EDRF, and increased adhesion molecules, cell damage, andmacrophage chemotaxis.

There are interactions between LUVs and small acceptors, such as HDL,apoprotein phospholipid complexes, and cyclodextrins. Liposomes remodelHDL into a better acceptor by donating extra phospholipid, and the smallacceptors act as a shuttle, carrying cholesterol efficiently from cellsto liposomes. LUVs do not elevate LDL concentrations and do not suppresshepatic LDL receptor gene expression. The medical utility for LUVsincludes restoring EDRF secretion by endothelial cells. High cholesterollevels inhibit endothelial release of EDRF not through cholesterol, butthrough an oxidized derivative of cholesterol. Because HDL itselfrestores EDRF release, perhaps through the removal of cholesterol or ofoxidized lipids, then liposomes would be able to do the same (the HDLferries cellular oxidized lipids to liposomes, for example).

The invention provides a method and mode of operation for modifyingcellular lipids, including oxidized lipids, without provoking a rise inLDL concentrations or harmfully disturbing hepatic homeostasis. Thus,the LUVs, presumably acting in concert with endogenous (or exogenous)small acceptors of cholesterol (such as HDL), pull oxidized lipids outof peripheral tissues and deliver them to the liver for disposal.Oxidized lipids have a wide range of harmful biological effects,including suppression of EDRF release, induction of cell adhesionmolecules, cellular damage, chemotaxis of macrophages, and so forth.

Oxidized lipids and their harmful effects include decrease endothelialC-type ANF; increased endothelial PAI-1 and decreased tPA and decreasedendothelial thrombomodulin. Liposomes enhance or participate in thiseffect. These changes impair the body's ability to dissolve clots. Themethods disclosed herein assist in ameliorating these harmful effects ofoxidized lipids. HDL acts in part by transporting enzymes thatinactivate biologically active oxidized lipids.

It is understood that oxidized LDL inhibits endothelial secretion ofC-type natrizuretic peptide (CNP). It is the lipid component of oxidizedLDL that mediates this effect. Most importantly, HDL blocks the actionof oxidized LDL, presumably by picking up oxidized lipids (e.g.,oxidized cholesterol). Coincubation with high-density lipoprotein (HDL),which alone had no effect on CNP release, significantly preventedOx-LDL-induced inhibition of CNP secretion by endothelial cells (ECs).Analysis by thin-layer chromatography demonstrated that oxysterols,including 7-ketocholesterol, in Ox-LDL were transferred from Ox-LDL toHDL during coincubation of these two lipoproteins. These resultsindicate that Ox-LDL, suppresses CNP secretion from ECs by7-ketocholesterol or other transferable hydrophilic lipids in Ox-LDL,and the suppressive effect of Ox-LDL is reversed by HDL.

Whatever molecule HDL picks up, the presence of liposomes or otheracceptors around as described herein will allow it to do a better job,because of remodeling of HDL by liposomes & shuttling of oxidized lipidsby HDL from tissues to liposomes (i.e., the liposomes continuously stripthe HDL). Liposomes with an exogenous small acceptor will also work.

It is further understood that transferable lipids in oxidizedlow-density lipoprotein stimulate plasminogen activator inhibitor-1 andinhibit tissue-type plasminogen activator release from endothelialcells. As above, it is the lipids in oxidized LDL, such as oxidizedforms of cholesterol, that produce the effect. It is understood thatoxidized low density lipoprotein reduced thrombomodulin transcription incultured human endothelial cells. It is appreciated that oxidized lipidsplay a role in atherosclerosis, and enzymes on HDL that inactivateoxidized lipids may contribute to a protective effect. It iscontemplated that the methods and compositions disclosed herein willhelp this proposed mechanism as well, for example, by removingend-products of these enzymes, by otherwise altering HDL, and byproviding an additional platform for enzyme transport and action.

As such the use of large liposomes to remove harmful lipids in general(here, oxidized lipids) from peripheral tissues, either directly or viaHDL, which would extract the lipids first, possibly inactivate them,then deliver them or their break-down products to liposomes in thecirculation is described. Direct methods to assess oxidation andoxidative damage in vivo include for lipids, assays for 8-epiPGF₂ alpha;for DNA, assess 8-oxo-2' deoxyguanosine; generally assess anti-oxidantenzymes in tissues; and assess anti-oxidants levels, such as vitamin E,vitamin C, urate, and reduced/oxidized glutathione.

Methods relating to and modes for effecting the reverse lipid transport,from cells, organs, & tissues, including transport of extracellularmaterial, and any exchangable material in general are described herein.This covers not just cholesterol, but also sphingomyelin, oxidizedlipids, lysophophatidylcholine, proteins, and also phospholipiddonation. Some effects of oxidized material include increasedcalcification in arterial cells as described above and below.

Three potential differences between large versus small liposome toexplain their different effects on LDL and apoB levels include:fenestral penetration (LUV<<SUV); rate of clearance (LUV<SUV, so thatLUVs produce a slow, sustained cholesterol delivery to the liver thatmay be less disruptive); and protein adsorption (LUV<<SUV).

Unesterfied cholesterol increases tissue factor expression bymacrophages. This is extremely important, because it ismacrophage-derived tissue factor that makes the material released byunstable, rupturing plaques such a powerful stimulus for a clot to formthat then blocks the vessel leading to a heart attack. The methods andmodes of operation and compositions of the invention act upon theexpression of tissue factor.

Poor absorption of proteins by large liposomes affects LDL levels and/oratherosclerosis by the following mechanisms: 1) acquisition of apoE fromVLDL by small liposomes impairs the removal of VLDL from thecirculation, thereby allowing it to be more efficiently converted intoatherogenic LDL; ii) absorbed proteins on small liposomes direct theseparticles into the wrong metabolic pool within the liver. Polyacrylamidegel electrophoresis shows that liposomes (actually small liposomes)increase the size of LDL. Liposomes are used to alter LDL size,composition and structure to decrease its atherogenicity.

Other properties of LDL could be changed by administration of liposomes.For example, liposomes reduce surface unesterified cholesterol; reducesurface sphingomyelin; replace surface phospholipids with POPC which ispoorly oxidized; supplement the LDL with antioxidants that were added tothe liposomes before administration. These changes would substantiallyalter arterial entry, retention, modification and atherogenicity of LDL.

The side-effects controlled are focused on hepatic cholesterolmetabolism, hepatic expression of genes involved in cholesterolmetabolism, and plasma concentrations of cholesterol-rich atherogeniclipoproteins that contain apolipoprotein B (chiefly, LDL). Reversetransport of sphingomyelin, for example, changes hepatic cholesterolmetabolism (cellular sphingomyelin affects the intracellulardistribution of cholesterol, and hence its regulatory effects; alsosphingomyelin is a precursor to ceramide, which mediates intracellularsignaling), though large liposomes appear to avoid any problems in thearea. The same holds true for reverse transport of oxidized forms ofcholesterol (they are even more potent that unoxidized cholesterol insuppressing LDL receptor gene expression). Cyclodextrins do not pick upphospholipids.

Liposomes pick up any exchangeable lipid (actually, any exchangeableamphipathic or hydrophobic material, which includes lipid or protein oranything else with these characteristics). This includes sphingomyelin,oxidized or modified lipids, such as oxidized sterols and phospholipids.Typically, such liposomes can pick up unesterified cholesterol and otherexchangeable material from other lipid bilayers, such as cell membranes,and from lipoproteins. Liposomes also pick up proteins and donatephospholipids. During and after these modifications, the liposomes areremoved from the plasma, chiefly by the liver. Throughout thisapplication, we will refer to this general process as "reverse lipidtransport", although it is understood that any exchangeable material intissues, blood, or liposomes could participate. Specific examples ofexchangeable material include unesterified cholesterol, oxidized formsof cholesterol, sphingomyelin, and other hydrophobic or amphipathicmaterial.

These molecules accumulate in atherosclerosis and mediate harmfuleffects (e.g., cholesterol, oxidized cholesterol, and other material,such as lysophospholipids) or in aging (e.g., sphingomyelin). Forexample, oxidized lipids, particularly sterols, alter many peripheraltissue functions, including stimulating calcification by arterial cellsin atherosclerosis & stimulating endothelial plasminogen activatorinhibitor-1 release by endothelial cells; other oxidized lipid productsinclude lysophospholipids that stimulate endothelial expression ofadhesion molecules that attract macrophages into lesions, andsphingomyelin accumulates in some cell-culture models of aging and, withcholesterol, may account for some of the cellular changes. Otherchanged, such as oxidation, may also mediate or accelerate aging. Manyof these molecules have been shown to be picked up by liposomes in vitro(e.g., cholesterol, sphingomyelin, & probably oxidized cholesterol) andmany by HDL (cholesterol, oxidized cholesterol by liposomes) but it islikely that they pick up these other molecules as well. In terms oftotal mass, however, the bulk of the acquired material is unesterifiedcholesterol, with proteins in second place. Alternatively, by acquiringunesterified cholesterol, the liposomes may reduce the amount ofoxidized cholesterol that develops, because there will be less startingmaterial.

The effective periods of time described herein should not be interpretedto exclude very long courses of treatment, lasting years, for example.Nor should it exclude repeated courses of treatment separated by weeks,months, or years.

Side effects include overload of the liver with cholesterol or othermaterials acquired by the liposomes; with subsequent alterations inhepatic function, such as suppression of LDL receptors, stimulation ofintrahepatic cholesterol esterification, stimulation of intrahepaticcholesterol esterification, stimulation of hepatic secretion ofatherogenic lipoproteins that contain apolipoprotein-B, and impaireduptake of atherogenic lipoproteins by the liver from plasma.

As used herein the word, "endogenous" indicates that the HDL arises fromwithin the body, and is not itself administered. HDL and relatedacceptors can, however, be administered.

The data indicates another difference between large and small liposomesin vivo. Before injection, the liposomes that are used in ourexperiments were essentially electrically neutral. indicated by afailure to migrate rapidly through a gel of agarose when an electricfield is applied. (This does not imply that charged liposomes or otherparticles could not be used. The small liposomes pick up proteins andother material, and become electrically charged: they now rapidlymigrate through agarose gels when an electric field is applied. Agarosegels of plasma samples we had stored from the three groups of rabbitswere run. The small liposomes became more mobile LDL in these gels. Thelarge liposomes were substantially less mobile, indicating a lowercharge density, reflecting a lower protein content.

Two explanations for the difference between large and small liposomesexist: 1) small ones penetrate through hepatic endothelial fenestraewhile large ones do not (thus, large ones go to Kupffer cells and smallones go to hepatic parenchymal cells and cause problems); 2) largeliposomes are known to be cleared by the liver somewhat more slowly thanare small liposomes (the reason is not known), and so may not overwhelmthe liver as easily. The data on charge density provides an explanationin part: less protein, therefore slower or altered hepatic uptake.

The delivery of cholesterol to the liver by LUVs is actually moreefficient than by SUVs, per mg of phospholipid. One difference is thatthe delivery by LUVs is steady over a long period after the injection,whereas the delivery by SUVs peaks then falls.

Some of the composition described herein include eggphosphatidylcholine; synthetic phosphatidylcholines that are notcrystalline at body temperature (e.g., they contain at least one doublebond) yet are resistant to oxidation (e.g., they do not have many doublebonds, such as 1-palmitoyl, 2-oleyl phosphatidylcholine, abbreviatedPOPC); other natural or synthetic phospholipids alone or in mixtures;any of the preceding supplemented or replaced with hydrophobic oramphipathic material that still allows a liposomal or micellarstructure. An extruder is certainly not the only conceivable method formaking large liposomes or even particularly LUVs. Other methods known topractioners in the field are available or can be adapted to make largeliposomes in general and LUVs in particular.

As used herein, a dose includes from 10 to 1600 mg of phospholipid, inthe form of large liposomes, per kg of body weight. Other acceptablerates described herein can be determined empirically by the response ofplasma LDL concentrations.

Where there is a change in membrane composition, as well as function,one can use an assay of membrane composition or an assay of tissuecomposition. Compositional assays should include lipids, proteins, andother components.

HDL can pick up oxidized material, and HDL-associated enzymes mayinactivate oxidized material.

The separations in time will depend on the actual dose of material, itseffects on hepatic cholesterol homeostasis, and whethercholesterol-lowering agents are being concurrently administered. Thus,for doses of about 300 mg of small liposomes per kg of body weight,slight disruptions will occur after even a single dose, and singleadministrations of higher doses may cause even more disruptions.Exemplary separations in time include one day to one month, but theprecise schedules would have to be determined by monitoring hepaticcholesterol metabolism and plasma levels od LDL and other atherogeniclipoproteins.

The major macrophages that would be involved in liposomal clearancewould be Kupffer cells in the liver and macrophages in the bone marrowor spleen. The catabolism here would be the so-called alternativepathway for initiating the conversion of cholesterol into bile acids(macrophages are known to have at least one cholesterol-catabolizingenzyme), or would be transfer of sterol (enzymatically altered or not)to other cells, such as hepatic parenchymal cells that would thendispose of the molecules.

The methods described herein also control effects of cellular aging.

The invention includes means for assessing the efficacy of liposomaltherapy by performing assays of oxidation in vitro and in vivo, assaysof oxidative susceptibility of plasma components, and assays of theability of altered HDL to inhibit oxidation (by binding oxidativeproducts and/or through its paroxinase or other anti-oxidantcomponents), and the ability of HDL or plasma or serum or blood tomobilize cholesterol and other exchangeable material.

Large liposomes may cause the mobilization of some material that istrapped between cells as well (this is the extracellular space). Thisextracellular material causes problems a) when it contacts cells orplatelets, altering their function and b) by simply taking up space.

Estimate rates of cholesterol mobilization can be empiricallydetermined. It is appreciated that the kinetics of liposomal clearanceis different in different species (the t_(1/2) of LUVs in mice is about8 h, but in rabbits it is about 24 th, and in humans it is longer).Thus, rates calculated may vary from species to species. Based on mydata on injection of 300 mg of SUVs into rabbits, the peak rate ofliposomal cholesterol removal from plasma was between 3 h and 6 h afterthe injection. At that point, the liposomes had raised plasmaunesterified cholesterol by just over 2 mmol/L; assuming a total plasmavolume of 90 mL in a 3-kg rabbit, the total liposomal cholesterol atthat point was 180 umoles; the t_(1/2) for SUVs in these rabbits wasabout 20 h, so roughly 10% is removed in 3 h; thus, the peak rate ofliposomal cholesterol removal was about 2 umoles/h/kg, and this caused asubsequent rise in plasma cholesteryl ester concentrations. Notice thatat other time periods after the injection, the rate of liposomalcholesterol removal from plasma was less. Note also that the liver isthe predominant organ for clearance, but not the sole organ forclearance.

It has been calculated that a single injection of 300 mg LUVs/kg into20-22-g mice mobilized about 2400 nmoles of cholesterol in the first 24h after injection. In contrast to the data with SUVs in rabbits, themobilization of cholesterol during the first 24 h in the mice injectedwith LUVs was quite steady. This calculates to about 4.7 μmoles/h/kgover this first 24-h period, which is actually more than the abovefigure of 2 μmoles/h/kg, which was a peak rate. It is not faircomparison, because the clearance of LUVs in mice is three times as fastas in rabbits. If we take 4.7 divided by 3, we get 1.6 umoles/h/kg,which is less than 2, but these are imperfect estimates. Human rates canbe empirically determined. It is clear, however, that LUVs deliver theircholesterol at a steady rate, whereas SUVs make a brief, rapid push oflipid into the liver.

At body temperature, the most desirable liposomes are fluid within theconfines of the bilayer, which is called the liquid crystalline state.Less desirable are liposomes in the gel state, which is less fluid.

It is understood that unesterified cholesterol stimulates macrophages toexpress more tissue factor, a substance known to provoke blood clots.This explains the presence of abundant tissue factor in rupture-proneplaques, which, when they rupture, expose tissue factor to plasma andprovoke a clot that can occlude the vessel, causing a heart attack. Thiswould be another example of an abnormal cellular function that may bereversed by removal of cholesterol by liposomes.

Several human conditions are characterized by distinctive lipidcompositions of tissues, cells, membranes and/or extracellular regions.For example, in atherosclerosis, cholesterol (unesterified, esterified,and oxidized forms) and other lipids accumulated in cells and inextracellular areas of the arterial wall and elsewhere. These lipidshave potentially harmful biologic effects, for example, by changingcellular functions and by narrowing the vessel lumen, obstructing theflow of blood. Removal of the lipids would provide numerous, substantialbenefits. Moreover, cells, membranes, tissues and extracellularstructures would benefit from composition and alteration that includeincreasing resistance to oxidation and oxidative damages, such as byincreasing the content and types of anti-oxidants, removing oxidizedmaterial, and increasing the content of material that is resistant tooxidation. In aging, cells have been shown to accumulate sphingomyelinand cholesterol, which alter cellular functions. These functions can berestored in vitro by removal of these lipids and replacement withphospholipid from liposomes. A major obstacle to performing similarlipid alterations in vivo has been disposition of the lipids mobilizedfrom tissues, cells, extracellular areas, and membranes. Natural (e.g.,high-density lipoproteins) and synthetic (e.g., small liposomes)particles that could mobilize peripheral tissue lipids have asubstantial disadvantage: they delivery their lipids to the liver in amanner that disturbs hepatic cholesterol homeostasis, resulting inelevations in plasma concentrations of harmful lipoproteins, such aslow-density lipoprotein (LDL), a major atherogenic lipoprotein.

The invention described herein provides methods and compositions relatedto the "reverse" transport of cholesterol and other materials andcompounds from peripheral tissues to the liver in vivo while controllingplasma LDL concentration.

Agarose gel electrophoreses of plasma samples from the last a set ofrabbits injected with LUVs, SUVs, or saline (these agarose gels separateparticles by their charge, which is not the same from one type ofparticle to another) were performed. Freshly made SUVs migrate veryslowly through agarose, which indicates that freshly made liposomes havevery little charge. After injection into animals or after co-incubationwith plasma or lipoproteins, SUVs pick up proteins from lipoproteins.These proteins give more charge to the SUVs and substantially enhancetheir migration through agarose gels. SUVs after exposure to plasmamigrate faster through these gels than does LDL.

The gels showed a substantial difference between LUVs and SUVs. Asexpected, the SUVs migrated ahead of LDL in these gels. The LUVs,however, migrated almost exactly where freshly made, protein-freeliposomes migrate. This result indicates that LUVs, unlike SUVs, do notreadily pick up proteins from circulating lipoproteins.

There is a direct verification of this difference between the liposomes.Human HDL (which has most of the proteins that liposomes pick up) wasincubated with either LUVs or SUVs, then the liposomes were reisolated,and assayed their protein-to-phospholipid ratios. Per amount ofliposomal phospholipid, the SUVs picked up about 40 times as muchprotein as did the LUVs. This difference appears to arise because of thedifference in surface curvature: SUVs are smaller, so their surface ismore tightly curved, thus under greater strain, proteins can more easilyinsert.

There are three most likely metabolic effects of the difference inprotein uptake between the two types of liposomes are as follows:

1. VLDL has two metabolic fates: it can be removed from plasma before itis fully converted to LDL by lipolytic enzymes, or it can be fullyconverted into circulating LDL. SUVs strip apoE off VLDL, therebyslowing its clearance from plasma and favoring its conversion to LDL. Incontrast, LUVs leave apoE on VLDL, and so LDL concentrations in plasmawould not rise.

2. Absorbed apoproteins might play a role in directing liposomes todifferent hepatic metabolic pools.

Here are some ways to assay effect on oxidation in vivo: Catella F,Reilly M P, Delanty N. Lawson J A, Moran N, Meagher E, FitzGerald G A.Physiological formation of 8-epi-PGF2 alpha in vivo is not affected bycyclooxygenase inhibition. Adv Prostaglandin Thromboxane Leukot Res.23:233-236, 1995. These authors describes 8-epi-PGF₂ alpha, which is anend-product of lipid oxidation. This molecule can be used, they suggest,as a measure of lipid oxidative flux in an animal. It is superior toother commonly used measure of oxidation in vivo, such as antioxidantlevels (which are affected by diet), thiobarituric acid reactivesubstances (some sugars interfere with this assay), and short-livedoxidative intermediates (these do not indicate total flux of materialbeing oxidized). Administration of LUVs, by removing oxidized lipidsfrom the periphery, would might lower total oxidative flux in vivo, and8-epi-PGF₂ alpha would be a suitable way to measure this; Cadet J,Ravanat J L, Buchko G W, Yeo H C, Ames B N. Singlet oxygen DNA damage:chromatographic and mass spectrometric analysis of damage products.Methods Enzymol. 234:79-88, 1994. they describe 8-oxo-2'-deoxyguanosine,which is an end-product of DNA oxidation. As above, this molecule can beused as a measure of DNA oxidative flux in an animal. Administration ofLUVs would lower DNA oxidative flux in vivo, and this is a suitable wayto measure this; and, Xia E, Rao G, Van Remmen H, Heydari A R,Richardson A. Activities of antioxidant enzymes in various tissues ofmale Fischer 344 rats are altered by food restriction. J Nutr.125(2):195-201, 1995. Antioxidant enzymes in tissues were measured, toindicate de-oxidant capacity. LUVs help this. Anti-oxidant levels(vitamin E, ascorbate, urate); oxidized and reduced glutathione; andmany other measures can be used to assess peripheral oxidation andoxidative damage. Again, these and other measures would be coupled withLUV administration, to assess efficacy of the therapy.

Other particles that mimic there properties of large liposomes will actsimilarly, to mobilize peripheral lipids and other exchangeablematerials, and deliver exchangeable materials, while avoiding harmfuldisruptions in hepatic cholesterol homeostasis. For example, these wouldinclude emulsion particles that are two large to penetrate hepaticendothelial fenestrae, of a composition and structure that is taken upby the liver slowly, and/or a composition and structure that does notreadily acquire specific endogenous proteins. Such emulsions could bemade with or without proteins, and could be made from phospholipid and aneutral lipid, such as triglycerides or another neutral lipid.Angioplasty can of course be done on any blocked vessels, including butnot limited to femoral arteries. Vessels upon which angioplasty can bedone include, by way of example, arteries and veins. Exemplary arteriesinclude vesical arteries, an iliac artery, an umbilical artery, aninternal pudendal artery, an anterior humeral circumflex artery, acerebral artery, an intercostal artery, a spinal artery, a temporalartery, a tibial artery, an ulnar recurrent artery, a coratid artery,etc. Exemplary veins include an ascending lumbar vein, an angular vein,a facial vein, a jugular vein, a tibial vein, etc.

While only a few, preferred embodiments of the invention have beendescribed hereinabove, those of ordinary skill in the art will recognizethat the embodiment may be modified and altered without departing fromthe central spirit and scope of the invention. Thus, the preferredembodiment described hereinabove is to be considered in all respects asillustrative and not restrictive, the scope of the invention beingindicated by the appended claims, rather than by the foregoingdescription, and all changes which come within the meaning and range ofequivalency of the claims are intended to be embraced herein.

I claim:
 1. An improved method of angioplasty or cardiac catheterizationin which said improvement comprises:administering a therapeuticallyeffective amount of a lipid acceptor to a subject, said lipid acceptorbeing selected from the group consisting of large liposomes comprised ofphospholipids substantially free of sterol and small acceptors.
 2. Themethod in accordance with claim 1 in which said administration of saidlipid acceptor occurs at or over an effective period of time.
 3. Themethod in accordance with claim 2 in which said effective period of timeis in the range of about 1 minute to about two years from the time ofsaid angioplasty or said cardiac catheterization.
 4. The method inaccordance with claim 1 in which said administration of said lipidacceptor occurs simultaneously with said angioplasty or said cardiaccatheterization.
 5. The method of claim 1 further comprising assessing avascular function.
 6. The method of claim 5 in which the step ofassessing a vascular function is selected from the group consisting ofmeasuring blood flow in a carotid artery, measuring blood flow in alower limb, measuring blood flow in a vessel using ultrasound, measuringblood flow using an MRI, measuring blood flow using a radioisotopetracer, and measuring blood viscosity.
 7. The method of claim 1 in whichsaid angioplasty is performed on a blocked vessel.
 8. The method ofclaim 7 in which said blocked vessel is selected from the groupconsisting a femoral vessel and a coronary vessel.
 9. The method ofclaim 1 in which said angioplasty comprises carotid endarterectomy. 10.The method of claim 1 in which said angioplasty comprises surgery torelieve a carotid obstruction.
 11. The method of claim 1 in which saidangioplasty comprises a mechanical or a surgical intervention to improveblood flow.
 12. The method in accordance with claim 11 in which saidintervention comprises placement of a stent.
 13. The method of claim 1in which said angioplasty comprises coronary angioplasty, femoralangioplasty, and a surgery to relieve a carotid obstruction.
 14. Themethod in accordance with claim 1 in which said large liposomes havediameters larger than about 120 nm.
 15. The method in accordance withclaim 1 in which the large liposomes have diameters larger than about125 nm.
 16. The method in accordance with claim 1 in which the largeliposomes have diameters larger than about 150 nm.
 17. The method inaccordance with claim 1 in which the large liposomes have diameterslarger than about 160 nm.
 18. The method in accordance with claim 1 inwhich the large liposomes have diameters larger than about 175 nm. 19.The method in accordance with claim 1 in which the large liposomes havediameters larger than about 200 nm.
 20. The method of claim 1 in whichsaid large liposomes have an unesterified cholesterol to phospholipidmolar ratio below about 1:10.
 21. The method of claim 1 in which saidlarge liposomes comprise POPC.
 22. The method of claim 1 in which thelarge liposomes are given in repeated doses.
 23. The method of claim 1in which said phospholipids are given in a daily dosage of less thanabout 600 mg/kg body weight.
 24. The method of claim 23 in which saiddosage is administered repeatedly.
 25. The method in accordance withclaim 1 in which the large liposomes are made of phospholipids selectedfrom the group consisting of phosphatidyl choline, phosphatidylglycerol, palmitoyl-oleoyl phosphatidyl choline, and combinationsthereof.
 26. The method in accordance with claim 1 further comprisingadministering a lipid lowering drug.
 27. The method in accordance withclaim 1 further comprising administering a drug that inhibitsrestenosis.
 28. The method in accordance with claim 1 further comprisinginserting a device in a subject that inhibits restenosis.
 29. The methodin accordance with claim 28 in which said device comprises a stent.